Azadirachtin
Azadirachtin A (112) was the first member of this group of highly oxygenated C-seco
meliacins, isolated by Butterworth and Morgan29 from neem seeds using a feeding inhibition
test against desert locust, Schistocerca gregaria. It caused 100% inhibition of feeding when
used at a concentration of 40μg L-' or 1 ng cm-2 when impregnated into a filter paper: In addition
to possessing antifeedant property, it also produced developmental abnormalities in almost all-
insect orders31. Its insect controlling activities have been extensively reviewed from time to time.
Azadirachtin A (112) was the first member of this group of highly oxygenated C-seco
meliacins, isolated by Butterworth and Morgan29 from neem seeds using a feeding inhibition
test against desert locust, Schistocerca gregaria. It caused 100% inhibition of feeding when
used at a concentration of 40μg L-' or 1 ng cm-2 when impregnated into a filter paper: In addition
to possessing antifeedant property, it also produced developmental abnormalities in almost all-
insect orders31. Its insect controlling activities have been extensively reviewed from time to time.
Structure elucidation of azadirachtin became as much challenging and formidable as was the
earlier task with nimbin (90). In fact, the same story was repeating here also. Three schools were
actively engaged in this stupendous effort and first hand accounts 0f their work appeared as a
symposia-in-print in Tetrahedron 152. It must be stated that the structure elucidation of
azadirachtin by nmr spectroscopy became a difficult task chiefly because of the following facts:
the molecule contains 16 oxygen atoms which dilute the spin-spin coupling values, its nmr
spectra are temperature-dependent indicating conformational flexibility of the molecule which
further complicates the interpretation of NOE results, it could not be obtained in a crystalline form,
owing probably to co-occurrence of its analogues. The preparative hplc was not employed by
early workers for its very detailed purification.
earlier task with nimbin (90). In fact, the same story was repeating here also. Three schools were
actively engaged in this stupendous effort and first hand accounts 0f their work appeared as a
symposia-in-print in Tetrahedron 152. It must be stated that the structure elucidation of
azadirachtin by nmr spectroscopy became a difficult task chiefly because of the following facts:
the molecule contains 16 oxygen atoms which dilute the spin-spin coupling values, its nmr
spectra are temperature-dependent indicating conformational flexibility of the molecule which
further complicates the interpretation of NOE results, it could not be obtained in a crystalline form,
owing probably to co-occurrence of its analogues. The preparative hplc was not employed by
early workers for its very detailed purification.
Initially, Morgan20 could determine its correct molecular formula, identify most of the functional groups and assign the partial structure.
NakanishiI63 in 1975 made a landmark in the elucidation of its complex structure (112a) by means of application of PRFT/CWD 13C NMR,
which brought out the structural similarity with salannin (85). Infra-red studies disclosed the presence of an intra-molecularly hydrogen
bonded hydroxyl (3465 cm-I), a free OH (3580 cm-I ) and highly hindered hydroxyl (3380 em-I) groups. The nature of the 35 carbons was
elucidated by a combination of cmr techniques i.e. proton noise decoupling (PND), continuous wave decoupling (CWD or off resonance
decoupling), partially relaxed Fourier transform (PRFT) and combined PRFT/CWD. Nakanishi made two corrections of earlier functional
group analysis- ¬the positions of acetate and tiglate groups were reversed and the second acetate persumed was actually C-18 methyl
appearing at exceptionally low field. Indeed, it was the first application of this spectroscopic technique in the structure elucidation of a
natural Product. However, there were some doubts about the nature of the ether linkages.
NakanishiI63 in 1975 made a landmark in the elucidation of its complex structure (112a) by means of application of PRFT/CWD 13C NMR,
which brought out the structural similarity with salannin (85). Infra-red studies disclosed the presence of an intra-molecularly hydrogen
bonded hydroxyl (3465 cm-I), a free OH (3580 cm-I ) and highly hindered hydroxyl (3380 em-I) groups. The nature of the 35 carbons was
elucidated by a combination of cmr techniques i.e. proton noise decoupling (PND), continuous wave decoupling (CWD or off resonance
decoupling), partially relaxed Fourier transform (PRFT) and combined PRFT/CWD. Nakanishi made two corrections of earlier functional
group analysis- ¬the positions of acetate and tiglate groups were reversed and the second acetate persumed was actually C-18 methyl
appearing at exceptionally low field. Indeed, it was the first application of this spectroscopic technique in the structure elucidation of a
natural Product. However, there were some doubts about the nature of the ether linkages.
After ten years, Ley and his co-workers21 submitted evidence in favour of structure (ll2b). The significant differences are (i) the tetrahydro
furan bridge is now between C-19 and C-11 (ii) the configuration at C-13 has been inversed. The evidences in favour of this revision were
the Overhauser effects observed between H-19a and H-1, H-7 and C-30 Me, H-15 and C-30 Me and that between H-7 and H-21. The
absence of an NOE between H-19b and H-16a and/or H-15 was also inconsistent with Nakanishi structure. Further, smaller NOE
enhancements supporting this revision included one from H-19a and H-2b and another from C-30 Me and H-21 and negative
enhancements due to 3-spin effects from H-19b to H-l and from H-19a to C-30 Me. Assignments of three hydroxy protons were supported
by additional NOE measurements carried out at 270 K. Enhancements ofH-9, H-5 and H-21 were attributed to proximity of OH¬13, OH-7
and OH-20 when these hydroxyl groups were irradiated. The chemical shift of OH-13 had a significantly smaller temperature and solvent
dependence than the OH-7 and OH-20 protons thereby implying its hindered nature.
furan bridge is now between C-19 and C-11 (ii) the configuration at C-13 has been inversed. The evidences in favour of this revision were
the Overhauser effects observed between H-19a and H-1, H-7 and C-30 Me, H-15 and C-30 Me and that between H-7 and H-21. The
absence of an NOE between H-19b and H-16a and/or H-15 was also inconsistent with Nakanishi structure. Further, smaller NOE
enhancements supporting this revision included one from H-19a and H-2b and another from C-30 Me and H-21 and negative
enhancements due to 3-spin effects from H-19b to H-l and from H-19a to C-30 Me. Assignments of three hydroxy protons were supported
by additional NOE measurements carried out at 270 K. Enhancements ofH-9, H-5 and H-21 were attributed to proximity of OH¬13, OH-7
and OH-20 when these hydroxyl groups were irradiated. The chemical shift of OH-13 had a significantly smaller temperature and solvent
dependence than the OH-7 and OH-20 protons thereby implying its hindered nature.
Independently, Kraus73 came out with an altogether different revision of its structure (112), which is the one now currently accepted. His
group concurred with the location of Oxygen Bridge between C-11 and C-19. The major significant revisions are the discovery of the
presence of an epoxide between C-13 and C-14, relocation of hydroxyl group at C-ll as a hemiacetal and confirmation of α-orientation of C-
13 Me. The additional evidences in favour of these significant and break-through revisions were the observed NOE on H-6, H-7, H-15 and ll-
OH due to saturation of the signal at δ 1.74 (C-30 Me) in addition to a positive effect on δ 4.15 (l9-Hb) but a negative effect on δ 3.63 (l9-Ha).
There was no effect observed in the signals of I-H and 2-Hb which one should expect if the signal at δ 1.74 would correspond to 19-H. The
IH and 13C nmr signals for H-7 and H-15, C-7 and C-15 were reversed on the basis of 13C_1H homodecoupling and 2D heteroscalar
correlation experiments respectively. Long-range coupling (COLOC experiment) was observed for C-7/30-H, C-ll/19-Ha, C-ll/ll-OH, C-13/18-
H, C-14/18-H, C-14/30-H and C-15/21-H. The identification of an epoxide and the location of hydroxyl groups were based on the 13C-
deuterium isotope shift experiments enabling the confirmation of 7-0H and 20-0H. However, a distinct isotope effect was observed on C-ll
(δ 104.07/103.97) but not on C-14 (0 70.39). Thus the 13-0H must indeed be ll-OH. The NOE measurements carried out by Ley and Morgan
are also in support of 11-0H rather than 13-0H. The chemical shifts of C-ll, C-13 and C-14 readily conform to the literature reports
respectively for a hemiacetal carbon and quarternary oxirane carbons. On the basis of the observed strong NOE between 7-H/21-H and 7-
0H/21-H and the chemical shift of 16-Ha, the epoxide group was assigned β-configuration.
group concurred with the location of Oxygen Bridge between C-11 and C-19. The major significant revisions are the discovery of the
presence of an epoxide between C-13 and C-14, relocation of hydroxyl group at C-ll as a hemiacetal and confirmation of α-orientation of C-
13 Me. The additional evidences in favour of these significant and break-through revisions were the observed NOE on H-6, H-7, H-15 and ll-
OH due to saturation of the signal at δ 1.74 (C-30 Me) in addition to a positive effect on δ 4.15 (l9-Hb) but a negative effect on δ 3.63 (l9-Ha).
There was no effect observed in the signals of I-H and 2-Hb which one should expect if the signal at δ 1.74 would correspond to 19-H. The
IH and 13C nmr signals for H-7 and H-15, C-7 and C-15 were reversed on the basis of 13C_1H homodecoupling and 2D heteroscalar
correlation experiments respectively. Long-range coupling (COLOC experiment) was observed for C-7/30-H, C-ll/19-Ha, C-ll/ll-OH, C-13/18-
H, C-14/18-H, C-14/30-H and C-15/21-H. The identification of an epoxide and the location of hydroxyl groups were based on the 13C-
deuterium isotope shift experiments enabling the confirmation of 7-0H and 20-0H. However, a distinct isotope effect was observed on C-ll
(δ 104.07/103.97) but not on C-14 (0 70.39). Thus the 13-0H must indeed be ll-OH. The NOE measurements carried out by Ley and Morgan
are also in support of 11-0H rather than 13-0H. The chemical shifts of C-ll, C-13 and C-14 readily conform to the literature reports
respectively for a hemiacetal carbon and quarternary oxirane carbons. On the basis of the observed strong NOE between 7-H/21-H and 7-
0H/21-H and the chemical shift of 16-Ha, the epoxide group was assigned β-configuration.
X-ray diffraction studies with crystalline detigloyldihydroazadirachtin27 confirmed the Kraus structure. The highly hindered nature of the 11-
0H was reasoned to be due to the existence of an intramolecular hydrogen bonding between C-ll hydroxy and the epoxy oxygen (2.66 A °,O-
H O angle 154°). The intramolecular hydrogen bonding between C-20 and C-7 hydroxyl oxygen atoms (3.13 AO, O-H…..O angle 149°)
was rather weak. These two hydrogen bonds are stated to act as main stabilizing factors for the structure. The mass spectral
interpretations in the light of the revised structure were also proposed22.
0H was reasoned to be due to the existence of an intramolecular hydrogen bonding between C-ll hydroxy and the epoxy oxygen (2.66 A °,O-
H O angle 154°). The intramolecular hydrogen bonding between C-20 and C-7 hydroxyl oxygen atoms (3.13 AO, O-H…..O angle 149°)
was rather weak. These two hydrogen bonds are stated to act as main stabilizing factors for the structure. The mass spectral
interpretations in the light of the revised structure were also proposed22.
Apparently contradicting results have been put forth on the nature of hydroxyl groups. While Ley and co-workers22 have shown the
existence of a strong hydrogen bonding between ll-OH and epoxide oxygen both by X-ray crystallography and nmr studies on temperature
dependency of the chemical shifts of the three hydroxyl groups Nakanishi and co-workersl54 produced evidence to show that azadirachtin
has a free rotation around C-8 and C-14 single bond and exists as two rotamers at 180 K. According to them, II-OH is the least hydrogen
bonded (hindered) and hence the facile formation of monoacetate (ll-O-acetyl). Kraus et al.,78 also observed the presence of two rotamers
in the ratio of 3:2 at 183K as well as the distinct NOE's between protons of ring Band D, thereby supporting certain restriction of the rotation
around C-8/C-14 bond.
existence of a strong hydrogen bonding between ll-OH and epoxide oxygen both by X-ray crystallography and nmr studies on temperature
dependency of the chemical shifts of the three hydroxyl groups Nakanishi and co-workersl54 produced evidence to show that azadirachtin
has a free rotation around C-8 and C-14 single bond and exists as two rotamers at 180 K. According to them, II-OH is the least hydrogen
bonded (hindered) and hence the facile formation of monoacetate (ll-O-acetyl). Kraus et al.,78 also observed the presence of two rotamers
in the ratio of 3:2 at 183K as well as the distinct NOE's between protons of ring Band D, thereby supporting certain restriction of the rotation
around C-8/C-14 bond.
Table 9. C-secomeliacins of azadirachtin group isolated mostly from seed kernels
Stru- Name Substituents Molecular m.p. Ref.
ture R1 R2 R3 X Formula Weight (*C) No.
No.
112. Azadirachtin A Tg Ac COOMe α-COOMe C35H44O16 720 165 Seed oil
β-OH
113. 3-Desacetyl-3-cinnamoyl
azadirachtin Tg Cin COOMe α-COOMe C42H48O16 808 78
β-OH
114. Azadirachtin B H Tg COOMe α-COOMe C33H42O14 662 204-6 80,67,117
β-H
115. Azadirachtin D Tg Ac Me α-COOMe C34H44O14 676 118
β-OH
116. Azadirachtin E Tg Ac COOMe α-COOMe C30H38O15 638 - 118
β-OH
117. Azadirachtin H Tg Ac COOMe -H, -OH C33H42O14 662 248 55,56
118. Azadirachtin I Tg Ac Me -H, -OH C32H42O12 618 200 55,56
119. Vepaol OMe H - - C36H48O17 762 73.122
120. Isovepaol H OMe - - C36H48O17 762 122
121. Azadirachtin F H Tg H, OH - C33H44O14 664 - 118
122. 1,3-Diacetyl-11,19-
deoxa-11oxo-meliacarp Ac Ac O - C31H40O13 620 Amorph. 79
123. Azadirachtin G C33H42O14 662 - 118
124. Azadirachtin K C34H40O15 688 260 57
125. 11-Methoxy-azadirachtinin C36H46O16 734 Amorph. 78
ture R1 R2 R3 X Formula Weight (*C) No.
No.
112. Azadirachtin A Tg Ac COOMe α-COOMe C35H44O16 720 165 Seed oil
β-OH
113. 3-Desacetyl-3-cinnamoyl
azadirachtin Tg Cin COOMe α-COOMe C42H48O16 808 78
β-OH
114. Azadirachtin B H Tg COOMe α-COOMe C33H42O14 662 204-6 80,67,117
β-H
115. Azadirachtin D Tg Ac Me α-COOMe C34H44O14 676 118
β-OH
116. Azadirachtin E Tg Ac COOMe α-COOMe C30H38O15 638 - 118
β-OH
117. Azadirachtin H Tg Ac COOMe -H, -OH C33H42O14 662 248 55,56
118. Azadirachtin I Tg Ac Me -H, -OH C32H42O12 618 200 55,56
119. Vepaol OMe H - - C36H48O17 762 73.122
120. Isovepaol H OMe - - C36H48O17 762 122
121. Azadirachtin F H Tg H, OH - C33H44O14 664 - 118
122. 1,3-Diacetyl-11,19-
deoxa-11oxo-meliacarp Ac Ac O - C31H40O13 620 Amorph. 79
123. Azadirachtin G C33H42O14 662 - 118
124. Azadirachtin K C34H40O15 688 260 57
125. 11-Methoxy-azadirachtinin C36H46O16 734 Amorph. 78
In addition to azadirachtin, neem oil elaborate several analogues. Remboldl18,119 was among the first to report the co-occurrence of
isomeric azadirachtins. For this reason, the original compound is known as azadirachtin A. So far, thirteen more analogues/derivatives
have been reported from neem (Table 9). Azadirachtin B (114) is identical with Kraus's 3-tigloylazadirachtol67 as well as Kubo's
deacetylazadirachtinol8O. Earlier Ley and his group22 claimed to have isolated I-tigloylazadirachtol but later reconciled it to be azadirachtin
B.23 Only partial structure could be assigned to azadirachtin C. The detigloylderivative of 112 is azadirachtin E (116) while azadirachtin D
(115) is the C-29 methyl analogue of 112. In 115, the C-29 COOMe group is replaced by methyl. Azadirachtin F (121) contains a free II-OH
and C-19 Me in place of the ether bridge between C-19 and C-I1. The novel meliacarpin derivative79 (122) is the II-oxo isomer of 121. Both
of them can be considered possible precursors of azadirachtin-A. Azadirachtins H (117) and I (118) differ from azadirachtin A respectively
due to loss of C-12 and reduction of C-29 COOMe to Me55.56. Biogenetically, 118 may be an immediate precursor of 112. Azadirachtin G
(123) is 13,14-deoxyazadirachtin B and the recently discovered azadirachtin K57 (124) contains a α-keto-δ-Iactone between C-19 and C-12
in place of the five membered lactol ring. Interestingly, the C-12 carboxyl is found lactonised with C-19 in 124. There is no information on
azadirachtin J. Sankaram et al.,122 announced the isolation of C-23 epimers, 22,23-dihydro-23-methoxyazadirachtins and named them as
vepaol (119) and isovepaol (120). Vepaol, the 23-β-methoxy epimer is synonymous with the one reported by Kraus.73 l-Tigloyl-3-acetyl-ll-
methoxyazadirachtinin (125) was isolated from neem bare8. Later, 119 and 120 were synthetically obtained from azadirachtin91. 3-
Deacetyl-3-cinnamoylazadirachtin 78 (113) was found in leaves while others in seeds.
isomeric azadirachtins. For this reason, the original compound is known as azadirachtin A. So far, thirteen more analogues/derivatives
have been reported from neem (Table 9). Azadirachtin B (114) is identical with Kraus's 3-tigloylazadirachtol67 as well as Kubo's
deacetylazadirachtinol8O. Earlier Ley and his group22 claimed to have isolated I-tigloylazadirachtol but later reconciled it to be azadirachtin
B.23 Only partial structure could be assigned to azadirachtin C. The detigloylderivative of 112 is azadirachtin E (116) while azadirachtin D
(115) is the C-29 methyl analogue of 112. In 115, the C-29 COOMe group is replaced by methyl. Azadirachtin F (121) contains a free II-OH
and C-19 Me in place of the ether bridge between C-19 and C-I1. The novel meliacarpin derivative79 (122) is the II-oxo isomer of 121. Both
of them can be considered possible precursors of azadirachtin-A. Azadirachtins H (117) and I (118) differ from azadirachtin A respectively
due to loss of C-12 and reduction of C-29 COOMe to Me55.56. Biogenetically, 118 may be an immediate precursor of 112. Azadirachtin G
(123) is 13,14-deoxyazadirachtin B and the recently discovered azadirachtin K57 (124) contains a α-keto-δ-Iactone between C-19 and C-12
in place of the five membered lactol ring. Interestingly, the C-12 carboxyl is found lactonised with C-19 in 124. There is no information on
azadirachtin J. Sankaram et al.,122 announced the isolation of C-23 epimers, 22,23-dihydro-23-methoxyazadirachtins and named them as
vepaol (119) and isovepaol (120). Vepaol, the 23-β-methoxy epimer is synonymous with the one reported by Kraus.73 l-Tigloyl-3-acetyl-ll-
methoxyazadirachtinin (125) was isolated from neem bare8. Later, 119 and 120 were synthetically obtained from azadirachtin91. 3-
Deacetyl-3-cinnamoylazadirachtin 78 (113) was found in leaves while others in seeds.
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